Project Summary: The long-term objective of Dr. Sinn is to pursue an academic career at a major research university studying the biology and development of lentiviral vectors. This will be achieved with the aid of Drs. McCray and Voytas and the support of the Dept. of Pediatrics at the University of Iowa. The immediate interest of Dr. Sinn is the general safety and utility of lentiviral vectors for use in a broad range of gene therapy applications, such as cystic fibrosis. Gene therapy for cystic fibrosis, a disease that has pulmonary and digestive manifestations, is directly applicable to the mission of the NIDDK. To persistently expressa therapeutic transgene, a retroviral vector must integrate into a host cell chromosome. This critical process makes lentiviral vectors an attractive tool to achieve life-long gene delivery; however, the nonspecific nature of retroviral integration presents inherent hazards and variations in gene expression. If integration could be restricted to preferred genomic loci, the safety and utility of lentiviral vectors would be vastly improved. In this proposal a novel strategy is investigated to modify the integrase (IN) gene of a feline immunodeficiency virus (FIV)-based lentiviral vector to achieve restricted integration. A "tethering" model is proposed in which an engineered hybrid protein that contains a protein binding domain and a DNA binding domain will direct the lentiviral vector integration complex to preferred sites on chromosomal DNA. Three aims are proposed: 1) determine the capacity to which FIV IN can be modified and still retain its function, as determined by titering assays as well as in vitro catalytic and integrase assays; 2) confirm high affinity protein-protein interactions of modified FIV IN to the tethering protein by yeast two-hybrid assays and demonstrate that these complexes can mediate restricted integration in a cell-system. These studies will focus the ultimate goal to, 3) map integration sites of the modified vector into genomic DNA and demonstrate a restricted pattern of integration in those cells in which the tethering protein is co-expressed. Relevance: Lentiviral vectors have the potential to persistently correct genetic diseases. However, such vectors integrate nonspecifically into the host genome and therefore present a risk of disrupting normal gene function at the site of insertion. Successful site-restricted lentiviral vector integration into human genomic DNA would have exciting and broad applications in the gene therapy field.